The ever-increasing global demand for energy has led to depletion of fossil fuels, which are buried combustible geologic deposits of organic materials that have been converted to crude oil, coal, natural gas, or heavy oils. Because fossil fuels were formed by exposure to heat and pressure in the earth's crust over hundreds of millions of years, they are a finite, non-renewable resource. Further, the burning of fossil fuels is thought to play a key role in global warming. Accordingly, there is a need for non-fossil fuel energy sources.
Hydrocarbons from biological sources represent a cleaner, sustainable alternative energy source. Further, many industries, including plastics and chemical manufacturers, rely heavily on the availability of hydrocarbons for manufacturing processes. Currently, energy-rich lipids and fatty acids (“nature's petroleum”) are isolated from plant and animal oils to produce diverse products such as fuels and oleochemicals. Recent efforts have focused on the microbial production of fatty acids and fatty acid derivatives by cost-effective bioprocesses. Methods of producing fatty acids and/or fatty acid derivatives in microbial hosts are described in, e.g., PCT Publication Nos. WO 2007/136762, WO 2008/119082, WO 2009/009391, WO 2009/076559, WO 2009/111513, WO 2010/006312, WO 2010/044960, WO 2010/118410, WO 2010/126891, WO 2011/008535 and WO 2011/019858 and in Schirmer et al., Science 329(5991):559-562 (2010).
Free fatty acids are known to cause damage to cellular membranes and are thus difficult to produce in amounts sufficient for large scale production. The reduction of fatty acids to more neutral lipids such as wax esters may help to circumvent free fatty acid toxicity. Wax esters possess high energy density relative to shorter-chain biofuel products such as ethanol, and can be produced in cultured cells via a series of enzymatic processes. Wax esters have numerous commercial applications in, e.g., the medical, cosmetic and dietetic industries. For example, wax esters may be used as components of candles, cosmetics, lubricants, printing inks, solvents and fuels.
Wax esters, which have an ‘A’ chain, derived from a fatty alcohol, and a ‘B’ chain, derived from an acyl-thioester molecule, i.e., acyl-CoA, are produced by a condensation reaction between a fatty acyl-thioester substrate and a fatty alcohol, catalyzed by a wax ester synthase. Wax ester synthases have been identified in, e.g., Acinetobacter (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002); Kalscheuer and Steinbuchel, J. Biol. Chem. 278:8075-8082 (2003); Kalscheuer et al., Appl. Environ. Microbiol. 72:1373-1379 (2006)), Marinobacter (Holtzapple and Schmidt-Dannert, J. Bacteriol. 189:3804-3812 (2007)), Arabidopsis (Li et al., Plant Physiol. 148:97-107 (2008)), petunia (King et al., Planta 226:381-394 (2007)), jojoba (Lardizabal et al., Plant Physiol. 122:645-655 (2000), and mammalian species (Cheng and Russell, J. Biol. Chem. 279:37798-37807 (2004); Yen et al., J. Lipid Res. 46:2388-2397 (2005)).
Fatty acid esters, which are the product of a condensation reaction between an acyl-CoA molecule and an alcohol of any chain length, can also be produced by wax ester synthases. For example, a fatty acid ester can be the condensation product of methanol, ethanol, propanol, butanol, isobutanol, 2-methylbutanol, 3-methylbutanol, or pentanol with an acyl-CoA molecule. In some instances, fatty acid esters such as fatty acid methyl esters (“FAME”) or fatty acid ethyl esters (“FAEE”) can be produced by supplying the alcohol used in the reaction (e.g., methanol or ethanol) to the culture media. Similarly, wax esters can be produced by supplying fatty alcohols (e.g., hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, etc.) to the culture medium of a host microorganism that expresses a wax synthase.
If wax esters are to be produced entirely by a host microorganism, however, the host microorganism must produce a fatty alcohol substrate. Enzymes that convert fatty acyl-thioesters to fatty alcohols or fatty aldehydes are commonly known as fatty acyl reductases (“FARs”). FARs have been identified in, e.g., Euglena (see, e.g., Teerawanichpan et al., Lipids 45:263-273 (2010)), Arabidopsis (see, e.g., Rowland et al., Plant Physiol. 142:866-877 (2006), Doan et al., J. Plant Physiol. 166:787-796 (2009) and Domergue et al., Plant Physiol. 153:1539-1554 (2010)), Artemisia (see, e.g., Maes et al., New Phytol. 189:176-189 (2011)), jojoba (see, e.g., Metz et al., Plant Physiol. 122:635-644 (2000)), moth (see, e.g., Lienard et al., Proc. Natl. Acad. Sci. 107:10955-10960 (2010)), bee (see, e.g., Teerawanichpan et al., Insect Biochemistry and Molecular Biology 40:641-649 (2010)) and in mammals (see, e.g., Honsho et al., J. Biol. Chem. 285:8537-8542 (2010)). Certain alcohol-forming acyl-CoA reductases are thought to generate fatty alcohols directly from acyl-CoA. Enzyme-based conversion of acyl-CoA to fatty alcohol can also occur in a two-enzyme, two-step reaction; in the first step, acyl-CoA is reduced to fatty aldehyde by an aldehyde-forming acyl-CoA reductase, and in the second step, the fatty aldehyde is reduced to a fatty alcohol by a fatty aldehyde reductase.
Typically, to produce a fatty acid ester or wax ester in a microorganism, it is necessary for the cell to produce various enzymes in addition to a wax ester synthase, and, where a wax ester is being produced entirely by the cell, an alcohol-forming reductase that generates the fatty alcohol substrate. For example, in a host that does not endogenously produce acyl-CoA, it may be necessary to introduce, e.g., a gene encoding a fatty acyl thioesterase to convert acyl-acyl carrier protein (acyl-ACP) to free fatty acids and a gene encoding an acyl-CoA synthetase to convert free fatty acids to acyl-CoA. For example, cyanobacteria do not produce acyl-CoA, and the genomes of cyanobacterial species sequenced to date do not include genes encoding acyl-ACP thioesterases, acyl-CoA thioesterases, or acyl-CoA synthetases, as cyanobacterial genes originally annotated as encoding acyl-CoA synthetases have been demonstrated to encode acyl-ACP synthetases, used in fatty acid recycling (Kaczmarzyk and Fulda (2010) Plant Physiol. 152: 1598-1610). Gene(s) encoding a fatty acyl thioesterase and/or an acyl-CoA synthetase are also added to host organisms that naturally produce acyl-CoA, to ensure adequate levels of acyl-CoA for the production of wax esters. Introducing several heterologous pathway components, however, may lead to difficulties in appropriately balancing enzyme expression and activity to produce the desired wax ester end product in sufficiently high yields for large scale production. Moreover, the buildup of intermediates such as free fatty acids and fatty alcohols may be toxic to host cells.
Accordingly, there remains a need in the art for more scalable, efficient and economic methods for producing fatty acid esters and wax esters.